Novel esterases and uses thereof

ABSTRACT

The present invention relates to novel esterases, more particularly to esterase variants having improved activity and/or improved thermostability compared to the esterase of SEQ ID NO: 1 and the uses thereof for degrading polyester containing material, such as plastic products. The esterases of the invention are particularly suited to degrade polyethylene terephthalate, and material containing polyethylene terephthalate.

The present invention relates to novel esterases, more particularly toesterases having improved activity and/or improved thermostabilitycompared to a parent esterase. The present invention also relates touses of said novel esterases for degrading polyester containingmaterial, such as plastic products. The esterases of the invention areparticularly suited to degrade polyethylene terephthalate, andpolyethylene terephthalate containing material.

BACKGROUND

Esterases are able to catalyze the hydrolysis of a variety of polymers,including polyesters. In this context, esterases have shown promisingeffects in a number of industrial applications, including as detergentsfor dishwashing and laundry applications, as degrading enzymes forprocessing biomass and food, as biocatalysts in detoxification ofenvironmental pollutants or for the treatment of polyester fabrics inthe textile industry. The use of esterases as degrading enzymes forhydrolyzing polyethylene terephthalate (PET) is of particular interest.Indeed, PET is used in a large number of technical fields, such as inthe manufacture of clothes, carpets, or in the form of a thermoset resinfor the manufacture of packaging or automobile plastics, etc., so thatPET accumulation in landfills becomes an increasing ecological problem.

The enzymatic degradation of polyesters, and particularly of PET, isconsidered as an interesting solution to decrease plastic wasteaccumulation. Indeed, enzymes may accelerate hydrolysis of polyestercontaining material, and more particularly of plastic products, even upto the monomer level. Furthermore, the hydrolysate (i.e., monomers andoligomers) can be recycled as material for synthesizing new polymers.

In this context, several esterases have been identified as candidatedegrading enzymes for polyesters, and some variants of such esteraseshave been developed. Among esterases, cutinases, also known as cutinhydrolases (EC 3.1.1.74), are of particular interest. Cutinases havebeen identified from various fungi (P. E. Kolattukudy in “Lipases”, Ed.B. Borg-stróm and H. L. Brockman, Elsevier 1984, 471-504), bacteria andplant pollen. Recently, metagenomics approaches have led toidentification of additional esterases.

However, there is still a need for esterases with improved activityand/or improved thermostability compared to already known esterases, toprovide polyester degrading processes more efficient and thereby morecompetitive.

SUMMARY OF THE INVENTION

The present invention provides new esterases exhibiting increasedactivity and/or increased thermostability compared to a parent, orwild-type esterase, having the amino acid sequence as set forth in SEQID No 1. This wild-type esterase corresponds to the esterase referencedas E5BBQ3 in UniProt database and described as having a polyesterdegrading activity. The esterases of the present invention areparticularly useful in processes for degrading plastic products, moreparticularly plastic products containing PET.

In this regard, it is an object of the invention to provide an esterasewhich (i) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identityto the full length amino acid sequence set forth in SEQ ID No 1, and(ii) has at least one substitution at a position selected fromF209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F, D94S, S121W,T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M, G205C/K,T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A, P242K, G243Y, P244N,G247A/D/E/H/S, G250C/Y and P260S wherein the positions are numbered byreference to the amino acid sequence set forth in SEQ ID No 1, (iii) hasa polyester degrading activity. Preferably said esterase exhibits anincreased thermostability and/or an increased degrading activitycompared to the esterase of SEQ ID No 1.

Preferably, the esterase comprises at least one substitution selectedfrom T168Q, F209I/G/H/L/R/S/T and N212D/Q, more preferably selected fromT168Q, F209I and N212D, more preferably at least F209I.

It is another object of the invention to provide a nucleic acid encodingan esterase of the invention. The present invention also relates to anexpression cassette or an expression vector comprising said nucleicacid, and to a host cell comprising said nucleic acid, expressioncassette or vector.

The present invention also provides a composition comprising an esteraseof the present invention, a host cell of the present invention, orextract thereof.

It is a further object of the invention to provide a method of producingan esterase of the invention comprising:

-   -   (a) culturing the host cell according to the invention under        conditions suitable to express a nucleic acid encoding an        esterase; and optionally    -   (b) recovering said esterase from the cell culture.

It is a further object of the invention to provide a method of degradinga polyester comprising

-   -   (a) contacting the polyester with an esterase according to the        invention or a host cell according to the invention or a        composition according to the invention; and, optionally    -   (b) recovering monomers and/or oligomers.

Particularly, the invention provides a method of degrading PET,comprising contacting PET with at least one esterase of the invention,and optionally recovering monomers and/or oligomers of PET.

The invention also relates to the use of an esterase of the inventionfor degrading PET or a plastic product containing PET.

The present invention also relates to a polyester containing material inwhich an esterase or a host cell or a composition of the invention isincluded.

The present invention also relates to a detergent composition comprisingthe esterase or host cell according to the invention or a compositioncomprising an esterase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present disclosure will be best understood by reference to thefollowing definitions.

Herein, the terms “peptide”, “polypeptide”, “protein”, “enzyme” refer toa chain of amino acids linked by peptide bonds, regardless of the numberof amino acids forming said chain. The amino acids are hereinrepresented by their one-letter or three-letters code according to thefollowing nomenclature: A: alanine (Ala); C: cysteine (Cys); D: asparticacid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine(Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L:leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline(Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T:threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine(Tyr).

The term “esterase” refers to an enzyme which belongs to a class ofhydrolases classified as EC 3.1.1 according to Enzyme Nomenclature thatcatalyzes the hydrolysis of esters into an acid and an alcohol. The term“cutinase” or “cutin hydrolase” refers to the esterases classified as EC3.1.1.74 according to Enzyme Nomenclature that are able to catalyse thechemical reaction of production of cutin monomers from cutin and water.

The terms “wild-type protein” or “parent protein” refer to thenon-mutated version of a polypeptide as it appears naturally. In thepresent case, the parent esterase refers to the esterase having theamino acid sequence as set forth in SEQ ID No 1.

The terms “mutant” and “variant” refer to polypeptides derived from SEQID No 1 and comprising at least one modification or alteration, i.e., asubstitution, insertion, and/or deletion, at one or more (e.g., several)positions and having a polyester degrading activity. The variants may beobtained by various techniques well known in the art. In particular,examples of techniques for altering the DNA sequence encoding thewild-type protein, include, but are not limited to, site-directedmutagenesis, random mutagenesis and synthetic oligonucleotideconstruction. Thus, the terms “modification” and “alteration” as usedherein in relation to a particular position means that the amino acid inthis particular position has been modified compared to the amino acid inthis particular position in the wild-type protein.

A “substitution” means that an amino acid residue is replaced by anotheramino acid residue. Preferably, the term “substitution” refers to thereplacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues, rare naturallyoccurring amino acid residues (e.g. hydroxyproline, hydroxylysine,allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine,N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline,pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), andnon-naturally occurring amino acid residue, often made synthetically,(e.g. cyclohexyl-alanine). Preferably, the term “substitution” refers tothe replacement of an amino acid residue by another selected from thenaturally-occurring standard 20 amino acid residues (G, P, A, V, L, I,M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign “+” indicates acombination of substitutions. In the present document, the followingterminology is used to designate a substitution: L82A denotes that aminoacid residue (Leucine, L) at position 82 of the parent sequence issubstituted by an Alanine (A). A121V/I/M denotes that amino acid residue(Alanine, A) at position 121 of the parent sequence is substituted byone of the following amino acids: Valine (V), Isoleucine (I), orMethionine (M). The substitution can be a conservative ornon-conservative substitution. Examples of conservative substitutionsare within the groups of basic amino acids (arginine, lysine andhistidine), acidic amino acids (glutamic acid and aspartic acid), polaramino acids (glutamine, asparagine and threonine), hydrophobic aminoacids (methionine, leucine, isoleucine, cysteine and valine), aromaticamino acids (phenylalanine, tryptophan and tyrosine), and small aminoacids (glycine, alanine and serine).

Unless otherwise specified, the positions disclosed in the presentapplication are numbered by reference to the amino acid sequence setforth in SEQ ID No 1.

As used herein, the term “sequence identity” or “identity” refers to thenumber (or fraction expressed as a percentage %) of matches (identicalamino acid residues) between two polypeptide sequences. The sequenceidentity is determined by comparing the sequences when aligned so as tomaximize overlap and identity while minimizing sequence gaps. Inparticular, sequence identity may be determined using any of a number ofmathematical global or local alignment algorithms, depending on thelength of the two sequences. Sequences of similar lengths are preferablyaligned using a global alignment algorithm (e.g. Needleman and Wunschalgorithm; Needleman and Wunsch, 1970) which aligns the sequencesoptimally over the entire length, while sequences of substantiallydifferent lengths are preferably aligned using a local alignmentalgorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981)or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)).Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer softwareavailable on internet web sites such as http://blast.ncbi.nlm.nih.gov/or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, % amino acid sequenceidentity values refers to values generated using the pair wise sequencealignment program EMBOSS Needle that creates an optimal global alignmentof two sequences using the Needleman-Wunsch algorithm, wherein allsearch parameters are set to default values, i.e. Scoringmatrix=BLOSUM62, Gap open=11, Gap extend=1.

A “polymer” refers to a chemical compound or mixture of compounds whosestructure is constituted of multiple monomers (repeat units) linked bycovalent chemical bonds. Within the context of the invention, the termpolymer includes natural or synthetic polymers, constituted of a singletype of repeat unit (i.e., homopolymers) or of a mixture of differentrepeat units (i.e., copolymers or heteropolymers). According to theinvention, “oligomers” refer to molecules containing from 2 to about 20monomers.

In the context of the invention, a “polyester containing material” or“polyester containing product” refers to a product, such as plasticproduct, comprising at least one polyester in crystalline,semi-crystalline or totally amorphous forms. In a particular embodiment,the polyester containing material refers to any item made from at leastone plastic material, such as plastic sheet, tube, rod, profile, shape,film, massive block, etc., which contains at least one polyester, andpossibly other substances or additives, such as plasticizers, mineral ororganic fillers. In another particular embodiment, the polyestercontaining material refers to a plastic compound, or plasticformulation, in a molten or solid state, suitable for making a plasticproduct. In another particular embodiment, the polyester containingmaterial refers to textile, fabrics or fibers comprising at least onepolyester. In another particular embodiment, the polyester containingmaterial refers to plastic waste or fiber waste comprising at least onepolyester.

In the present description, the term “polyester(s)” encompasses but isnot limited to polyethylene terephthalate (PET), polytrimethyleneterephthalate (PTT), polybutylene terephthalate (PBT), polyethyleneisosorbide terephthalate (PEIT), polylactic acid (PLA),polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylenesuccinate adipate (PBSA), polybutylene adipate terephthalate (PBAT),polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethyleneadipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures ofthese polymers.

New Esterases

The present invention provides novel esterases with improved activityand/or improved thermostability compared to a parent esterase. Moreparticularly, the inventors have designed novel enzymes particularlysuited for use in industrial processes. The esterases of the inventionare particularly suited to degrade polyesters, more particularly PET,including PET containing material and particularly plastic productcontaining PET. In a particular embodiment, the esterases exhibit bothan increased activity and an increased thermostability.

It is therefore an object of the present invention to provide esterasesthat exhibit an increased activity, compared to the esterase having theamino acid sequence as set forth in SEQ ID No 1 also referenced asparent esterase.

Particularly, the inventors have identified specific amino acid residuesin SEQ ID No 1, which are intended to be in contact with a polymersubstrate in the X-ray crystal structure (i.e., folded 3D structure) ofthe esterases that may be advantageously modified to promote the contactof the substrate with the esterases and leading to an increasedadsorption of the polymer and/or thereby to an increased activity of theesterases on this polymer.

Within the context of the invention, the term “increased activity” or“increased degrading activity” indicates an increased ability of theesterase to degrade a polyester and/or an increased ability to adsorb ona polyester, at a given temperature as compared to the ability of theesterase of SEQ ID No 1 to degrade and/or adsorb on same polyester atsame temperature. Particularly, the esterase of the invention has anincreased PET degrading activity. Such an increase may be at least 10%greater than the PET degrading activity of the esterase of SEQ ID No 1,preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%,120%, 130% or greater. Particularly, the degrading activity is adepolymerization activity leading to monomers and/or oligomers of thepolyester, which can be further retrieved and optionally reused.

The “degrading activity” of an esterase may be evaluated by the oneskilled in the art, according to methods known per se in the art. Forinstance, the degrading activity can be assessed by measurement of thespecific polymer's depolymerization activity rate, the measurement ofthe rate to degrade a solid polymer compound dispersed in an agar plate,or the measurement of the polymer's depolymerization activity rate inreactor. Particularly, the degrading activity may be evaluated bymeasuring the “specific degrading activity” of an esterase. The“specific degrading activity” of an esterase for PET corresponds to μmolof PET hydrolyzed/min or mg of equivalent TA produced/hour and per mg ofesterase during the initial period of the reaction (i.e. the first 24hours) and is determined from the linear part of the hydrolysis curve ofthe reaction, such curve being set up by several samplings performed atdifferent time during the first 24 hours. As another example, the“degrading activity” may be evaluated by measuring, after a definedperiod of time, the rate and/or yield of oligomers and/or monomersreleased under suitable conditions of temperature, pH and buffer, whencontacting the polymer or the polymer-containing plastic product with adegrading enzyme.

The ability of an enzyme to adsorb on a substrate may be evaluated bythe one skilled in the art, according to methods known per se in theart. For instance, the ability of an enzyme to adsorb on a substrate canbe measured from a solution containing the enzyme and wherein the enzymehas been previously incubated with a substrate under suitableconditions.

The inventors have also identified target amino acid residues in SEQ IDNo 1, that may be advantageously modified to improve the stability ofcorresponding esterases at high temperatures (i.e., improvedthermostability), and advantageously at temperature above 50° C.,preferably 60° C., more preferably above 65° C.

It is therefore an object of the present invention to provide newesterases that exhibit increased thermostability as compared to thethermostability of the esterase having the amino acid sequence set forthin SEQ ID No 1 (i.e., the parent esterase).

Within the context of the invention, and unless otherwise specified, agiven temperature corresponds to said temperature+/−1° C.

Within the context of the invention, the term “increasedthermostability” indicates an increased ability of an esterase to resistto changes in its chemical and/or physical structure at hightemperatures, and particularly at temperature between 50° C. and 90° C.,as compared to the esterase of SEQ ID No 1. In a particular embodiment,the thermostability of the esterases is improved, as compared to thethermostability of the parent esterase, at temperature between and 90°C., between 50° C. and 80° C., between 50° C. and 75° C., between 50° C.and 70° C., between 50° C. and 65° C., between 55° C. and 90° C.,between 55° C. and 80° C., between 55° C. and between 55° C. and 70° C.,between 55° C. and 65° C., between 60° C. and 90° C., between 60° C. and80° C., between 60° C. and 75° C., between 60° C. and 70° C., between60° C. and 65° C., between 65° C. and 90° C., between 65° C. and 80° C.,between 65° C. and 75° C., between 65° C. and 70° C. In a particularembodiment, the thermostability of the esterases is improved, ascompared to the thermostability of the parent esterase, at least attemperature between 50° C. and 65° C.

Particularly, the thermostability may be evaluated through theassessment of the melting temperature (Tm) of the esterase. In thecontext of the present invention, the “melting temperature” refers tothe temperature at which half of the enzyme population considered isunfolded or misfolded. Typically, esterases of the invention show anincreased Tm of about 1° C., 2° C., 3° C., 4° C., 5° C., 10° C. or more,as compared to the Tm of the esterase of SEQ ID No 1. In particular,esterases of the present invention can have an increased half-life at atemperature between 50° C. and 90° C., as compared to the esterase ofSEQ ID No 1. Particularly, esterases of the present invention can havean increased half-life at temperature between 50° C. and 90° C., between50° C. and 80° C., between 50° C. and 75° C., between 50° C. and 70° C.,between 50° C. and between 55° C. and 90° C., between 55° C. and 80° C.,between 55° C. and 75° C., between and 70° C., between 55° C. and 65°C., between 60° C. and 90° C., between 60° C. and 80° C., between 60° C.and 75° C., between 60° C. and 70° C., between 60° C. and 65° C.,between 65° C. and between 65° C. and 80° C., between 65° C. and 75° C.,between 65° C. and 70° C., as compared to the esterase of SEQ ID No 1.In a particular embodiment, the esterases of the present invention havean increased half-life at least at temperature between 50° C. and 65°C., as compared to the esterase of SEQ ID No 1.

The melting temperature (Tm) of an esterase may be measured by the oneskilled in the art, according to methods known per se in the art. Forinstance, the DSF may be used to quantify the change in thermaldenaturation temperature of the esterase and thereby to determine itsTm. Alternatively, the Tm can be assessed by analysis of the proteinfolding using circular dichroism. Preferably, the Tm is measured usingDSF or circular dichroism as exposed in the experimental part. In thecontext of the invention, comparisons of Tm are performed with Tm thatare measured under same conditions (e.g. pH, nature and amount ofpolyesters, etc.).

Alternatively, the thermostability may be evaluated by measuring theesterase activity and/or the polyester depolymerization activity of theesterase after incubation at different temperatures and comparing withthe esterase activity and/or polyester depolymerization activity of theparent esterase. The ability to perform multiple rounds of polyester'sdepolymerization assays at different temperatures can also be evaluated.A rapid and valuable test may consist on the evaluation, by halodiameter measurement, of the esterase ability to degrade a solidpolyester compound dispersed in an agar plate after incubation atdifferent temperatures.

Thus, it is an object of the present invention to provide an esterasewhich (i) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identityto the full length amino acid sequence set forth in SEQ ID No 1, and(ii) has at least one amino acid substitution selected fromF209I/A/G/H/L/R/S/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F, D94S,S121W, A125G, L152Q, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P,E202M, G205C/K, T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A,P242K, G243Y, P244N, G247A/D/E/H/S, G250C/Y and P260S wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID No 1, (iii) has a polyester degrading activity, and preferably(iv) has an increased thermostability and/or an increased degradingactivity compared to the esterase of SEQ ID No 1.

In an embodiment, the esterase comprises at least one substitutionselected from F209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F,D94S, S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M,G205C/K, T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A, P242K,G243Y, P244N, G247A/D/E/H/S, G250C/Y and P260S.

Preferably, the esterase comprises at least one substitution selectedfrom D12F/Y, T50P, T63Q, S66H, W69R, T89R, D94S, S121W, A125G, L152Q,T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M, G205C, T207L,F209I, P211A, N212D, K216P, K220E, Q238T, L240A, P242K, G243Y, P244N,G247A, G250C/Y and P260S, more preferably selected from D12F/Y, T50P,T63Q, S66H, W69R, T89R, D94S, S121W, T153A, N158Q, T168Q, P180E, A182R,F188I/Y, S197P, E202M, G205C, T207L, F209I, P211A, N212D, K216P, K220E,Q238T, L240A, P242K, G243Y, P244N, G247A, G250C/Y and P260S.

In an embodiment, the esterase comprises at least one substitutionselected from T168Q, F209I/A/G/H/L/R/S/T and N212D/Q, preferablyselected from T168Q, F209I/G/H/L/R/T and N212D/Q, more preferablyselected from T168Q, F209I and N212D.

In a preferred embodiment, the esterase comprises at least onesubstitution selected from F209I/G/H/L/R/T and T168Q, preferablyselected from F209I and T168Q.

Particularly, the esterase comprises at least a substitution selectedfrom F209I/A/G/H/L/R/S/T, preferably selected from F209I/G/H/L/R/T, morepreferably the substitution F209I.

In an embodiment, the esterase further comprises at least onesubstitution at position selected from T11, D12, S23, T50, A53, Y60,T61, T63, A65, T88, T89, L90, Q92, M107, S121, A125, L152, M127, G135,5136, P151, L157, K159, T177, T183, D204, T207, F209, A210, N212, 1213,K216, Q238, D246, E253 and D174, preferably selected from T61, A65, Q92,G135, T177, T183, D204, F209, N212 and E253.

Particularly, the esterase further comprises at least one substitutionselected from T11N, D12H, S23P, T50E, A53L, Y60F/M, T61M/V, T63N, A65T,T88S, T89Q, L90W/F, Q92G/P, M107L, S121R, A125G, L152Q, M127V, G135A,S136T, P151A, L157E/G/N/Q/W/T, K159T, T177N/H/Q, T183E, D204C/K/R,T207D, F209W/S/A, A210T, N212M, 1213F, K216N, Q238D, D246Y/C/E/P, E253Cand D174R, preferably selected from T61M/V, A65T, Q92G/P, G135A,T177N/H/Q, T183E, D204C/K/R, F209W/S/A, N212M and E253C, more preferablyselected from T61M/V, A65T, Q92G/P, G135A, T177N, T183E, D204C, F209W,N212M and E253C.

In a preferred embodiment, the esterase further comprises at least onesubstitution selected from Q92G/P, G135A, T183E, D204C/K/R, F209W/S/Aand E253C, preferably selected from Q92G, G135A, T183E, D204C/K/R, F209Wand E253C. Particularly, the esterase further comprises at least onesubstitution selected from Q92G/P, G135A, T183E, D204C/K/R and E253C,preferably selected from Q92G, G135A, T183E, D204C and E253C.

In a preferred embodiment, the esterase further comprises at least thecombination of substitutions D204C+E253C.

In an embodiment, the esterase further comprises at least one amino acidsubstitution selected from D204K/R and at least the amino acid residueE253 as in the parent esterase.

In an embodiment, the esterase comprises a substitution selected fromF209I/A/G/H/L/R/S/T, preferably selected from F209I/G/H/L/R/T, and atleast one substitution selected from T61M/V, A65T, Q92G/P, G135A, T168Q,T177N/H/Q, T183E, D204C/K/R, N212D/Q/M and E253C, preferably selectedfrom Q92G/P, G135A, T168Q, T183E, D204C/K/R and E253C, more preferablyselected from Q92G, G135A, T168Q, T183E, D204C and E253C. Particularly,the esterase comprises a substitution selected from F209I/A/G/H/L/R/S/T,preferably selected from F209I/G/H/L/R/T, and at least twosubstitutions, preferably at least three substitutions selected fromT61M/V, A65T, Q92G/P, G135A, T168Q, T177N/H/Q, T183E, D204C/K/R,N212D/Q/M and E253C, preferably selected from Q92G/P, G135A, T168Q,T183E, D204C/K/R and E253C, more preferably selected from Q92G, G135A,T168Q, T183E, D204C and E253C.

Particularly, the esterase comprises at least three substitutions,preferably at least four substitutions selected fromF209I/A/G/H/L/R/S/T/W, T61M/V, A65T, Q92G/P, G135A, T168Q, T177N/H/Q,T183E, D204C/K/R, N212D/Q/M and E253C. Preferably, the esterasecomprises at least three substitutions, preferably at least foursubstitutions selected from F209I/G/H/L/R/T/W, T61M/V, A65T, Q92G/P,G135A, T168Q, T177N/H/Q, T183E, D204C/K/R, N212D/Q/M and E253C.

In a particular embodiment, the esterase has the amino acid sequence setforth in SEQ ID No 1 with one to thirty-one amino acid substitutions, ascompared to SEQ ID No 1, selected from the group consisting inF209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F, D94S, S121W,T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M, G205C/K,T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A, P242K, G243Y, P244N,G247A/D/E/H/S, G250C/Y and P260S, preferably selected from F209I,D12F/Y, T50P, T63Q, S66H, W69R, T89R, D94S, S121W, T153A, N158Q, T168Q,P180E, A182R, F188I/Y, S197P, E202M, G205C, T207L, P211A, N212D, K216P,K220E, Q238T, L240A, P242K, G243Y, P244N, G247A, G250C/Y and P260S, morepreferably selected from F209I, D12F/Y, T50P, T63Q, S66H, W69R, T89R,D94S, S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M,G205C, T207L, P211A, N212D, K216P, K220E, Q238T, L240A, P242K, G243Y,P244N, G247A, G250C/Y and P260S. Particularly, the esterase has theamino acid sequence set forth in SEQ ID No 1 with one to three aminoacid substitutions, as compared to SEQ ID No 1, selected from T168Q,F209I/G/H/L/R/T and N212D/Q, preferably selected from T168Q, F209I andN212D.

In an embodiment, the esterase has the amino acid sequence set forth inSEQ ID No 1 with one or two amino acid substitutions, as compared to SEQID No 1, selected from the group consisting in F209I/G/H/L/R/T andT168Q, preferably selected from F209I and T168Q.

In a particular embodiment, the esterase has the amino acid sequence setforth in SEQ ID No 1 with a single amino acid substitution, as comparedto SEQ ID No 1, selected from the group consisting in F209I/G/H/L/R/T,D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F, D94S, S121W, T153A, N158Q,T168Q, P180E, A182R, F188I/Y, S197P, E202M, G205C/K, T207L, P211A,N212D/Q, K216P, K220E, Q238T, L240A, P242K, G243Y, P244N, G247A/D/E/H/S,G250C/Y and P260S, preferably selected from F209I, D12F/Y, T50P, T63Q,S66H, W69R, T89R, D94S, S121W, A125G, L152Q, T153A, N158Q, T168Q, P180E,A182R, F188I/Y, S197P, E202M, G205C, T207L, P211A, N212D, K216P, K220E,Q238T, L240A, P242K, G243Y, P244N, G247A, G250C/Y and P260S, morepreferably selected from F209I, D12F/Y, T50P, T63Q, S66H, W69R, T89R,D94S, S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M,G205C, T207L, P211A, N212D, K216P, K220E, Q238T, L240A, P242K, G243Y,P244N, G247A, G250C/Y and P260S. Particularly, the esterase comprises asingle substitution selected from T168Q, F209I/G/H/L/R/T and N212D/Q, ascompared to the esterase of SEQ ID No 1, preferably selected from T168Q,F209I and N212D.

In an embodiment, the esterase has the amino acid sequence set forth inSEQ ID No 1 with a single amino acid substitution, as compared to SEQ IDNo 1, selected from the group consisting in F209I/G/H/L/R/T and T168Q,preferably selected from F209I and T168Q.

Particularly, the esterase has the amino acid sequence set forth in SEQID No 1 with a single amino acid substitution, as compared to SEQ ID No1, selected from F209I/G/H/L/R/T, preferably the substitution F209I.Advantageously the esterase exhibits an increased degrading activitycompared to the esterase of SEQ ID No 1.

It is also an object of the invention to provide an esterase which (i)has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to thefull length amino acid sequence set forth in SEQ ID No 1, and (ii) hasat least three amino acid substitutions at positions selected from F209,T61, A65, Q92, G135, T168, T177, T183, D204, N212 and E253 wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID No 1, (iii) has a polyester degrading activity, and preferably(iv) has an increased thermostability and/or an increased degradingactivity compared to the esterase of SEQ ID No 1. Preferably theesterase comprises at least four amino acid substitutions at positionsselected from F209, T61, A65, Q92, G135, T168, T177, T183, D204, N212and E253.

Preferably, the esterase comprises at least three substitutions, morepreferably at least four amino acid substitutions selected fromF209I/A/G/H/L/R/S/T/W, T61M/V, A65T, Q92G/P, G135A, T168Q, T177N/H/Q,T183E, D204C/K/R, N212D/Q/M and E253C, preferably selected fromF209I/G/H/L/R/T/W, Q92G/P, G135A, T168Q, T183E, D204C/K/R and E253C,more preferably selected from F209I/W, Q92G/P, G135A, T168Q, T183E,D204C and E253C, even more preferably selected from F209I, Q92G, G135A,T168Q, T183E, D204C, and E253C.

In an embodiment, the esterase comprises a substitution selected fromF209I/A/G/H/L/R/S/T/W, preferably selected from F209I/G/H/L/R/T, and atleast two substitutions selected from T61M/V, A65T, Q92G/P, G135A,T168Q, T177N/H/Q, T183E, D204C/K/R, N212D/Q/M and E253C, preferablyselected from Q92G/P, G135A, T168Q, T183E, D204C/K/R, and E253C, morepreferably Q92G, G135A, T168Q, T183E, D204C, and E253C. Particularly,the esterase comprises a substitution selected fromF209I/A/G/H/L/R/S/T/W, preferably selected from F209I/G/H/L/R/T, and atleast three substitutions selected from T61M/V, A65T, Q92G/P, G135A,T168Q, T177N/H/Q, T183E, D204C/K/R, N212D/Q/M and E253C, preferablyselected from Q92G/P, G135A, T168Q, T183E, D204C/K/R, and E253C, morepreferably selected from Q92G, G135A, T168Q, T183E, D204C, and E253C.

In an embodiment, the esterase comprises at least a combination ofsubstitutions at positions F209+D204+E253, preferably at least acombination of substitutions selected fromF209I/A/G/H/L/R/S/T/W+D204C+E253C, more preferably a combinationselected from F209I/W+D204C+E253C, even more preferably the combinationof substitutions F209I+D204C+E253C.

In an embodiment, the esterase comprises at least a combination ofsubstitutions at positions F209+D204+E253 and at least one substitutionat positions selected from T61, A65, Q92, G135, T168, T177, T183 andN212, preferably at positions selected from Q92, G135, T168, and T183.

Preferably, the esterase comprises at least a combination ofsubstitution selected from F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C,preferably selected from F209I/A/G/H/L/R/S/T+D204C/K/R+E253C, morepreferably selected from F209I+D204C+E253C. Particularly, the esterasefurther comprises at least one substitution at position selected fromT61, A65, Q92, G135, T168, T177, T183 and N212, preferably at least onesubstitution selected from T61M/V, A65T, Q92G/P, G135A, T168Q,T177N/H/Q, T183E and N212D/Q/M, more preferably selected from Q92G/P,G135A, T168Q and T183E, even more preferably selected from Q92G, G135A,T168Q and T183E. In an embodiment, the esterase comprises an amino acidsequence that consists of the amino acid sequence set forth in SEQ ID No1 with the combination of substitutionsF209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C, preferably selected fromF209I/A/G/H/L/R/S/T+D204C/K/R+E253C, more preferably selected fromF209I+D204C+E253C.

In an embodiment, the esterase comprises at least a combination ofsubstitutions at positions selected from the group consisting ofF209+D204+E253, F209+D204+E253+Q92, F209+D204+E253+N212,F209+D204+E253+Q92+G135+T168, F209+D204+E253+Q92+G135+T168+T183,F209+D204+E253+Q92+T183, or F209+D204+E253+Q92+T168+T183. Preferably,the esterase comprises at least a combination of substitutions selectedfrom F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+N212D/M/Q,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+G135A+T168Q,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+G135A+T168Q+T183E,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+T183E,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+T168Q+T183E, morepreferably selected from F209I/W+D204C+E253C,F209I/W+D204C+E253C+Q92G/P, F209I/W+D204C+E253C+N212D/M,F209I/W+D204C+E253C+Q92G/P+G135A+T168Q,F209I/W+D204C+E253C+Q92G/P+G135A+T168Q+T183E,F209I/W+D204C+E253C+Q92G/P+T183E,F209I/W+D204C+E253C+Q92G/P+T168Q+T183E, even more preferably selectedfrom F209I+D204C+E253C+Q92G, F209I+D204C+E253C+Q92G+T183E andF209I+D204C+E253C+Q92G+G135A+T168Q+T183E. Advantageously, this esteraseexhibits both an increased thermostability and an increased polyesterdegrading activity compared to the esterase of SEQ ID No 1. Preferablythe esterase exhibits both an increased thermostability and an increasedpolyester degrading activity compared to the esterase of SEQ ID No 1 ata temperature between 50° C. and 65° C., more preferably at 50° C.and/or at 65° C.

In an embodiment, the esterase comprises at least a combination ofsubstitution at positions F209+D204+E253+Q92, preferably a combinationof substitutions selected from F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P,more preferably F209I+D204C+E253C+Q92G.

In an embodiment, the esterase may further comprise at least onesubstitution at positions selected from L14, G62, R73, D85, T86, A179,A206, N215, 1217, G219, F239, R245, F249, E251, V252 and D174.Preferably, the substitution is selected from L14D/E, G62D/S,R73C/D/E/F/G/I/M/N/Q/S/V, D85A/E/F, T86E/S, A179C, A206D, N215C/D/E,I217Q, G219A/E, F239E, R245C/E, F249T, E251D/E/H/S, V252T.

In a particular embodiment, the esterase has the amino acid sequence ofthe esterase consists in the amino acid sequence as set forth in SEQ IDNo 1 with three to eleven substitutions, as compared to SEQ ID No 1,selected from F209I/A/G/H/L/R/S/T/W, T61M/V, A65T, Q92G/P, G135A, T168Q,T177N/H/Q, T183E, D204C/K/R, N212D/Q/M and E253C, preferably with threeto seven substitutions, as compared to SEQ ID No 1, selected fromF209I/A/G/H/L/R/S/T/W, Q92G/P, G135A, T168Q, T183E, D204C/K/R and E253C,more preferably selected from F209I/W, Q92G/P, G135A, T168Q, T183E,D204C and E253C, even more preferably selected from F209I, Q92G, G135A,T168Q, T183E, D204C, and E253C.

In a particular embodiment, the amino acid sequence of the esteraseconsists in the amino acid sequence as set forth in SEQ ID No 1 with acombination of substitutions, as compared to SEQ ID No 1, selected fromF209I/A/G/H/L/R/S/T/W+D204C+E253C,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P,F209I/A/G/H/L/R/S/T/W+D204C+E253C+N212D/M/Q,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+G135A+T168Q,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+G135A+T168Q+T183E,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+T183E,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+T168Q+T183E, more preferablyselected from F209I/W+D204C+E253C, F209I/W+D204C+E253C+Q92G/P,F209I/W+D204C+E253C+N212D/M, F209I/W+D204C+E253C+Q92G/P+G135A+T168Q,F209I/W+D204C+E253C+Q92G/P+G135A+T168Q+T183E,F209I/W+D204C+E253C+Q92G/P+T183E,F209I/W+D204C+E253C+Q92G/P+T168Q+T183E, even more preferablyF209I+D204C+E253C+Q92G, F209I+D204C+E253C+Q92G+T183E andF209I+D204C+E253C+Q92G+G135A+T168Q+T183E. Advantageously, this esteraseexhibits both an increased thermostability and an increased degradingactivity compared to the esterase of SEQ ID No 1. Preferably theesterase exhibits both an increased thermostability and an increasedpolyester degrading activity compared to the esterase of SEQ ID No 1 ata temperature between and 65° C., more preferably at 50° C. and/or at65° C.

In another embodiment, the amino acid sequence of the esterase consistsof the amino acid sequence set forth in SEQ ID No 1 with a combinationof substitutions, as compared to SEQ ID No 1, selected fromF209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C, preferably selected fromF209I/A/G/H/L/R/S/T+D204C/K/R+E253C, more preferably selected fromF209I+D204C+E253C.

In an embodiment, the esterase comprises at least one amino acid residueselected from D176, H208, S130, M131, C241, C259, G59, H129, G132, W155,I171, I178, P214, D174, as in the parent esterase, i.e. the esterase ofthe invention is not modified at one, two or all of these positions.

In an embodiment, the esterase comprises at least the amino acids D176,H208 and S130 forming the catalytic site of the esterase and/or theamino acids C241 and C259 forming disulphide bond as in the parentesterase. Preferably the esterase comprises a least a combinationselected from D176+H208+S130, C241+C259 and D176+H208+S130+C241+C259 asin the parent esterase. In an embodiment, the esterase comprises thecombination D176+H208+S130+C241+C259+M131+D174 as in the parentesterase.

Alternatively or in addition, the esterase of the invention comprises atleast one amino acid selected from G59, H129, G132, W155, I171, I178 andP214 as in the parent esterase. Preferably, the esterase comprises atleast one amino acid selected from I171 and I178 as in the parentesterase, more preferably at least the combination I171+I178 as in theparent esterase. In an embodiment, the esterase comprises thecombination I171+I178+G59+H129+G132+W155+P214 as in the parent esterase.

In an embodiment, the esterase comprises the combinationD176+H208+S130+C241+C259+I171+I178 as in the parent protease.

Polyester Degrading Activity of the Variant

It is an object of the invention to provide new enzymes having anesterase activity. In a particular embodiment, the enzyme of theinvention exhibits a cutinase activity.

In a particular embodiment, the esterase of the invention has apolyester degrading activity, preferably a polyethylene terephthalate(PET) degrading activity, and/or a polybutylene adipate terephthalate(PBAT) degrading activity and/or a polycaprolactone (PCL) degradingactivity and/or a polybutylene succinate (PBS) activity, more preferablya polyethylene terephthalate (PET) degrading activity, and/or apolybutylene adipate terephthalate (PBAT) degrading activity. Even morepreferably, the esterase of the invention has a polyethyleneterephthalate (PET) degrading activity.

Advantageously, the esterase of the invention exhibits a polyesterdegrading activity at least in a range of temperatures from 20° C. to90° C., preferably from 40° C. to 90° C., more preferably from 50° C. to90° C., even more preferably from 60° C. and 90° C. Particularly, theesterase of the invention exhibits a polyester degrading activity in arange of temperatures from 65° C. and 65° C. and 85° C., 65° C. and 80°C., 70° C. and 90° C., 70° C. and 85° C., 70° C. and 80° C. In aparticular embodiment, the esterase exhibits a polyester degradingactivity at 60° C. In a particular embodiment, the esterase exhibits apolyester degrading activity at 70° C. In a particular embodiment, thepolyester degrading activity is still measurable at a temperaturebetween 55° C. and 70° C. As exposed above, temperatures must beconsidered+/−1° C.

In a particular embodiment, the esterase of the invention has anincreased polyester degrading activity at a given temperature, comparedto the esterase of SEQ ID No 1, and more particularly at a temperaturebetween 40° C. and 90° C., more preferably between 50° C. and 90° C.

In a particular embodiment, the esterase has a polyester degradingactivity at 65° C. at least 5% higher than the polyester degradingactivity of the esterase of SEQ ID No 1, preferably at least 10%, 20%,50%, 100% or more.

In a particular embodiment, the esterase of the invention exhibits ameasurable esterase activity at least in a range of pH from 5 to 9,preferably in a range of pH from 6 to 9, more preferably in a range ofpH from 6.5 to 9, even more preferably in a range of pH from 6.5 to 8.

Nucleic Acids, Expression Cassette, Vector, Host Cell

It is a further object of the invention to provide a nucleic acidencoding an esterase as defined above.

As used herein, the term “nucleic acid”, “nucleic sequence,”“polynucleotide”, “oligonucleotide” and “nucleotide sequence” refer to asequence of deoxyribonucleotides and/or ribonucleotides. The nucleicacids can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can be insingle stranded form or in duplex form or a mixture thereof. It can beof recombinant, artificial and/or synthetic origin and it can comprisemodified nucleotides, comprising for example a modified bond, a modifiedpurine or pyrimidine base, or a modified sugar. The nucleic acids of theinvention can be in isolated or purified form, and made, isolated and/ormanipulated by techniques known per se in the art, e.g., cloning andexpression of cDNA libraries, amplification, enzymatic synthesis orrecombinant technology. The nucleic acids can also be synthesized invitro by well-known chemical synthesis techniques, as described in,e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.

The invention also encompasses nucleic acids which hybridize, understringent conditions, to a nucleic acid encoding an esterase as definedabove. Preferably, such stringent conditions include incubations ofhybridization filters at about 42° C. for about 2.5 hours in 2×SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in1×SSC/0.1% SDS at 65° C. Protocols used are described in such referenceas Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (CurrentProtocols in Molecular Biology (1989)).

The invention also encompasses nucleic acids encoding an esterase of theinvention, wherein the sequence of said nucleic acids, or a portion ofsaid sequence at least, has been engineered using optimized codon usage.

Alternatively, the nucleic acids according to the invention may bededuced from the sequence of the esterase according to the invention andcodon usage may be adapted according to the host cell in which thenucleic acids shall be transcribed. These steps may be carried outaccording to methods well known to one skilled in the art and some ofwhich are described in the reference manual Sambrook et al. (Sambrook etal., 2001).

Nucleic acids of the invention may further comprise additionalnucleotide sequences, such as regulatory regions, i.e., promoters,enhancers, silencers, terminators, signal peptides and the like that canbe used to cause or regulate expression of the polypeptide in a selectedhost cell or system.

The present invention further relates to an expression cassettecomprising a nucleic acid according to the invention operably linked toone or more control sequences that direct the expression of said nucleicacid in a suitable host cell.

The term “expression”, as used herein, refers to any step involved inthe production of a polypeptide including, but being not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The term “expression cassette” denotes a nucleic acid constructcomprising a coding region, i.e. a nucleic acid of the invention, and aregulatory region, i.e. comprising one or more control sequences,operably linked.

Typically, the expression cassette comprises, or consists of, a nucleicacid according to the invention operably linked to a control sequencesuch as transcriptional promoter and/or transcription terminator. Thecontrol sequence may include a promoter that is recognized by a hostcell or an in vitro expression system for expression of a nucleic acidencoding an esterase of the present invention. The promoter containstranscriptional control sequences that mediate the expression of theenzyme. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell. The control sequence may also be atranscription terminator, which is recognized by a host cell toterminate transcription. The terminator is operably linked to the3′-terminus of the nucleic acid encoding the esterase. Any terminatorthat is functional in the host cell may be used in the presentinvention. Typically, the expression cassette comprises, or consists of,a nucleic acid according to the invention operably linked to atranscriptional promoter and a transcription terminator.

The invention also relates to a vector comprising a nucleic acid or anexpression cassette as defined above.

As used herein, the terms “vector” or “expression vector” refer to a DNAor RNA molecule that comprises an expression cassette of the invention,used as a vehicle to transfer recombinant genetic material into a hostcell. The major types of vectors are plasmids, bacteriophages, viruses,cosmids, and artificial chromosomes. The vector itself is generally aDNA sequence that consists of an insert (a heterologous nucleic acidsequence, transgene) and a larger sequence that serves as the “backbone”of the vector. The purpose of a vector which transfers geneticinformation to the host is typically to isolate, multiply, or expressthe insert in the target cell. Vectors called expression vectors(expression constructs) are specifically adapted for the expression ofthe heterologous sequences in the target cell, and generally have apromoter sequence that drives expression of the heterologous sequencesencoding a polypeptide. Generally, the regulatory elements that arepresent in an expression vector include a transcriptional promoter, aribosome binding site, a terminator, and optionally present operator.Preferably, an expression vector also contains an origin of replicationfor autonomous replication in a host cell, a selectable marker, alimited number of useful restriction enzyme sites, and a potential forhigh copy number. Examples of expression vectors are cloning vectors,modified cloning vectors, specifically designed plasmids and viruses.Expression vectors providing suitable levels of polypeptide expressionin different hosts are well known in the art. The choice of the vectorwill typically depend on the compatibility of the vector with the hostcell into which the vector is to be introduced. Preferably, theexpression vector is a linear or circular double stranded DNA molecule.

It is another object of the invention to provide a host cell comprisinga nucleic acid, an expression cassette or a vector as described above.The present invention thus relates to the use of a nucleic acid,expression cassette or vector according to the invention to transform,transfect or transduce a host cell. The choice of the vector willtypically depend on the compatibility of the vector with the host cellinto which it must be introduced.

According to the invention, the host cell may be transformed,transfected or transduced in a transient or stable manner. Theexpression cassette or vector of the invention is introduced into a hostcell so that the cassette or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector. The term“host cell” also encompasses any progeny of a parent host cell that isnot identical to the parent host cell due to mutations that occur duringreplication. The host cell may be any cell useful in the production of avariant of the present invention, e.g., a prokaryote or a eukaryote. Theprokaryotic host cell may be any Gram-positive or Gram-negativebacterium. The host cell may also be an eukaryotic cell, such as ayeast, fungal, mammalian, insect or plant cell. In a particularembodiment, the host cell is selected from the group of Escherichiacoli, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces,Pichia, Vibrio or Yarrowia.

The nucleic acid, expression cassette or expression vector according tothe invention may be introduced into the host cell by any method knownby the skilled person, such as electroporation, conjugation,transduction, competent cell transformation, protoplast transformation,protoplast fusion, biolistic “gene gun” transformation, PEG-mediatedtransformation, lipid-assisted transformation or transfection,chemically mediated transfection, lithium acetate-mediatedtransformation, liposome-mediated transformation.

Optionally, more than one copy of a nucleic acid, cassette or vector ofthe present invention may be inserted into a host cell to increaseproduction of the variant.

In a particular embodiment, the host cell is a recombinantmicroorganism. The invention indeed allows the engineering ofmicroorganisms with improved capacity to degrade polyester containingmaterial. For instance, the sequence of the invention may be used tocomplement a wild type strain of a fungus or bacterium already known asable to degrade polyester, in order to improve and/or increase thestrain capacity.

Production of Esterase

It is another object of the invention to provide a method of producingan esterase of the invention, comprising expressing a nucleic acidencoding the esterase and optionally recovering the esterase.

In particular, the present invention relates to in vitro methods ofproducing an esterase of the present invention comprising (a) contactinga nucleic acid, cassette or vector of the invention with an in vitroexpression system; and (b) recovering the esterase produced. In vitroexpression systems are well-known by the person skilled in the art andare commercially available.

Preferably, the method of production comprises

-   -   (a) culturing a host cell that comprises a nucleic acid encoding        an esterase of the invention under conditions suitable to        express the nucleic acid; and optionally    -   (b) recovering said esterase from the cell culture.

Advantageously, the host cell is a recombinant Bacillus, recombinant E.coli, recombinant Aspergillus, recombinant Trichoderma, recombinantStreptomyces, recombinant Saccharomyces, recombinant Pichia, recombinantVibrio or recombinant Yarrowia.

The host cells are cultivated in a nutrient medium suitable forproduction of polypeptides, using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium, from commercial suppliers or preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection).

If the esterase is excreted into the nutrient medium, the esterase canbe recovered directly from the culture supernatant. Conversely, theesterase can be recovered from cell lysates or after permeabilisation.The esterase may be recovered using any method known in the art. Forexample, the esterase may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. Optionally, the esterase may be partially or totallypurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction to obtainsubstantially pure polypeptides.

The esterase may be used as such, in purified form, either alone or incombinations with additional enzymes, to catalyze enzymatic reactionsinvolved in the degradation and/or recycling of polyester(s) and/orpolyester containing material, such as plastic products containingpolyester. The esterase may be in soluble form, or on solid phase. Inparticular, it may be bound to cell membranes or lipid vesicles, or tosynthetic supports such as glass, plastic, polymers, filter, membranes,e.g., in the form of beads, columns, plates and the like.

Composition

It is a further object of the invention to provide a compositioncomprising an esterase, or a host cell of the invention, or extractthereof. In the context of the invention, the term “composition”encompasses any kind of compositions comprising an esterase or host cellof the invention.

The composition of the invention may comprise from 0.1% to 99.9%,preferably from 0.1% to 50%, more preferably from 0.1% to 30%, even morepreferably from 0.1% to 5% by weight of esterase, based on the totalweight of the composition. Alternatively, the composition may comprisebetween 5 and 10% by weight of esterase of the invention.

The composition may be liquid or dry, for instance in the form of apowder. In some embodiments, the composition is a lyophilisate.

The composition may further comprise excipients and/or reagents etc.Appropriate excipients encompass buffers commonly used in biochemistry,agents for adjusting pH, preservatives such as sodium benzoate, sodiumsorbate or sodium ascorbate, conservatives, protective or stabilizingagents such as starch, dextrin, arabic gum, salts, sugars e.g. sorbitol,trehalose or lactose, glycerol, polyethyleneglycol, polypropyleneglycol, propylene glycol, sequestering agent such as EDTA, reducingagents, amino acids, a carrier such as a solvent or an aqueous solution,and the like. The composition of the invention may be obtained by mixingthe esterase with one or several excipients.

In a particular embodiment, the composition comprises from 0.1% to99.9%, preferably from 50% to 99.9%, more preferably from 70% to 99.9%,even more preferably from 95% to 99.9% by weight of excipient(s), basedon the total weight of the composition. Alternatively, the compositionmay comprise from 90% to 95% by weight of excipient(s).

In a particular embodiment, the composition may further compriseadditional polypeptide(s) exhibiting an enzymatic activity. The amountsof esterase of the invention will be easily adapted by those skilled inthe art depending e.g., on the nature of the polyester to degrade and/orthe additional enzymes/polypeptides contained in the composition.

In a particular embodiment, the esterase of the invention is solubilizedin an aqueous medium together with one or several excipients, especiallyexcipients which are able to stabilize or protect the polypeptide fromdegradation. For instance, the esterase of the invention may besolubilized in water, eventually with additional components, such asglycerol, sorbitol, dextrin, starch, glycol such as propanediol, salt,etc. The resulting mixture may then be dried so as to obtain a powder.Methods for drying such mixture are well known to the one skilled in theart and include, without limitation, lyophilisation, freeze-drying,spray-drying, supercritical drying, down-draught evaporation, thin-layerevaporation, centrifugal evaporation, conveyer drying, fluidized beddrying, drum drying or any combination thereof.

In a particular embodiment, the composition is under powder form andcomprises esterase and a stabilizing/solubilizing amount of glycerol,sorbitol or dextrin, such as maltodextrine and/or cyclodextrine, starch,glycol such as propanediol, and/or salt.

In a particular embodiment, the composition of the invention comprisesat least one recombinant cell expressing an esterase of the invention,or an extract thereof. An “extract of a cell” designates any fractionobtained from a cell, such as cell supernatant, cell debris, cell walls,DNA extract, enzymes or enzyme preparation or any preparation derivedfrom cells by chemical, physical and/or enzymatic treatment, which isessentially free of living cells. Preferred extracts areenzymatically-active extracts. The composition of the invention maycomprise one or several recombinant cells of the invention or extractthereof, and optionally one or several additional cells.

In an embodiment, the composition consists or comprises a culture mediumof a recombinant microorganism expressing and excreting an esterase ofthe invention. In a particular embodiment, the composition comprisessuch culture medium lyophilized.

Uses of Esterase

It is a further object of the invention to provide methods using anesterase of the invention for degrading and/or recycling in aerobic oranaerobic conditions polyester, or polyester containing material. Theesterases of the invention are particularly useful for degrading PET andPET containing material.

It is therefore an object of the invention to use an esterase of theinvention, or corresponding recombinant cell or extract thereof, orcomposition for the enzymatic degradation of a polyester.

In a particular embodiment, the polyester targeted by the esterase isselected from polyethylene terephthalate (PET), polytrimethyleneterephthalate (PTT), polybutylene terephthalate (PBT), polyethyleneisosorbide terephthalate (PEIT), polylactic acid (PLA),polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylenesuccinate adipate (PBSA), polybutylene adipate terephthalate (PBAT),polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethyleneadipate) (PEA), polyethylene naphthalate (PEN) and blends/mixtures ofthese materials, preferably polyethylene terephthalate.

In a preferred embodiment, the polyester is PET, and at least monomers(e.g., monoethylene glycol or terephthalic acid), and/or oligomers(e.g., methyl-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl)terephthalate (BHET), 1-(2-Hydroxyethyl) 4-methyl terephthalate (HEMT)and dimethyl terephthalate (DMT)) are recovered.

It is also an object of the invention to use an esterase of theinvention, or corresponding recombinant cell or extract thereof, orcomposition for the enzymatic degradation of at least one polyester of apolyester containing material.

It is another object of the invention to provide a method for degradingat least one polyester of a polyester containing material, wherein thepolyester containing material is contacted with an esterase or host cellor extract thereof or composition of the invention, thereby degradingthe at least one polyester of a polyester containing material.

Advantageously, polyester(s) is (are) depolymerized up to monomersand/or oligomers.

Particularly, the invention provides a method for degrading PET of a PETcontaining material, wherein the PET containing material is contactedwith an esterase or host cell or composition of the invention, therebydegrading the PET.

In an embodiment, at least one polyester is degraded intorepolymerizable monomers and/or oligomers, which may be advantageouslyretrieved in order to be reused. The retrieved monomers/oligomers may beused for recycling (e.g., repolymerizing polyesters) or methanization.In a particular embodiment, at least one polyester is PET, andmonoethylene glycol, terephthalic acid, methyl-2-hydroxyethylterephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET),1-(2-Hydroxyethyl) 4-methyl terephthalate (HEMT) and/or dimethylterephthalate (DMT) are retrieved.

In an embodiment, polyester(s) of the polyester containing material is(are) fully degraded.

The time required for degrading a polyester containing material may varydepending on the polyester containing material itself (i.e., nature andorigin of the polyester containing material, its composition, shapeetc.), the type and amount of esterase used, as well as various processparameters (i.e., temperature, pH, additional agents, etc.). One skilledin the art may easily adapt the process parameters to the polyestercontaining material and the envisioned degradation time.

Advantageously, the degrading process is implemented at a temperaturecomprised between 20° C. and 90° C., preferably between 40° C. and 90°C., more preferably between 50° C. and 70° C. In a particularembodiment, the degrading process is implemented at 60° C. In anotherparticular embodiment, the degrading process is implemented at 65° C. Inanother particular embodiment, the degrading process is implemented at70° C. More generally, the temperature is maintained below aninactivating temperature, which corresponds to the temperature at whichthe esterase is inactivated (i.e., has lost more than 80% of activity ascompared to its activity at its optimum temperature) and/or therecombinant microorganism does no more synthesize the esterase.Particularly, the temperature is maintained below the glass transitiontemperature (Tg) of the targeted polyester.

Advantageously, the process is implemented in a continuous flow process,at a temperature at which the esterase can be used several times and/orrecycled.

Advantageously, the degrading process is implemented at a pH comprisesbetween 5 to 9, preferably in a range of pH from 6 to 9, more preferablyin a range of pH from 6.5 to 9, even more preferably in a range of pHfrom 6.5 to 8.

In a particular embodiment, the polyester containing material may bepretreated prior to be contacted with the esterase, in order tophysically change its structure, so as to increase the surface ofcontact between the polyester and the esterase.

It is another object of the invention to provide a method of producingmonomers and/or oligomers from a polyester containing material,comprising exposing a polyester containing material to an esterase ofthe invention, or corresponding recombinant cell or extract thereof, orcomposition, and optionally recovering monomers and/or oligomers.

Monomers and/or oligomers resulting from the depolymerization may berecovered, sequentially or continuously. A single type of monomersand/or oligomers or several different types of monomers and/or oligomersmay be recovered, depending on the starting polyester containingmaterial.

The method of the invention is particularly useful for producingmonomers selected from monoethylene glycol and terephthalic acid, and/oroligomers selected from methyl-2-hydroxyethyl terephthalate (MHET),bis(2-hydroxyethyl) terephthalate (BHET), 1-(2-Hydroxyethyl) 4-methylterephthalate (HEMT) and dimethyl terephthalate (DMT), from PET, and/orplastic product comprising PET.

The recovered monomers and/or oligomers may be further purified, usingall suitable purifying methods and conditioned in a re-polymerizableform.

Recovered repolymerizable monomers and/or oligomers may be reused forinstance to synthesize polyesters. Advantageously, polyesters of samenature are repolymerized. However, it is possible to mix the recoveredmonomers and/or oligomers with other monomers and/or oligomers, in orderfor instance to synthesize new copolymers. Alternatively, the recoveredmonomers may be used as chemical intermediates in order to produce newchemical compounds of interest.

The invention also relates to a method of surface hydrolysis or surfacefunctionalization of a polyester containing material, comprisingexposing a polyester containing material to an esterase of theinvention, or corresponding recombinant cell or extract thereof, orcomposition. The method of the invention is particularly useful forincreasing hydrophilicity, or water absorbency, of a polyester material.Such increased hydrophilicity may have particular interest in textilesproduction, electronics and biomedical applications.

It is a further object of the invention to provide a polyestercontaining material in which an esterase of the invention and/or arecombinant microorganism expressing and excreting said esterase is/areincluded. As an example, processes for preparing such polyestercontaining material including an esterase of the invention are disclosedin the patent applications WO2013/093355, WO2016/198650, WO2016/198652,WO2019/043145 and WO2019/043134.

It is thus an object of the invention to provide a polyester containingmaterial containing an esterase of the invention and/or a recombinantcell and/or a composition or extract thereof and at least PET. Accordingto an embodiment, the invention provides a plastic product comprisingPET and an esterase of the invention having a PET degrading activity.

It is thus another object of the invention to provide a polyestercontaining material containing an esterase of the invention and/or arecombinant cell and/or a composition or extract thereof and at leastPBAT. According to an embodiment, the invention provides a plasticproduct comprising PBAT and an esterase of the invention having a PBATdegrading activity.

It is thus another object of the invention to provide a polyestercontaining material containing an esterase of the invention and/or arecombinant cell and/or a composition or extract thereof and at leastPBS. According to an embodiment, the invention provides a plasticproduct comprising PBS and an esterase of the invention having a PBSdegrading activity.

It is thus another object of the invention to provide a polyestercontaining material containing an esterase of the invention and/or arecombinant cell and/or a composition or extract thereof and at leastPCL. According to an embodiment, the invention provides a plasticproduct comprising PCL and an esterase of the invention having a PCLdegrading activity.

Classically, an esterase of the invention may be used in detergent,food, animal feed, paper making, textile and pharmaceuticalapplications. More particularly, the esterase of the invention may beused as a component of a detergent composition. Detergent compositionsinclude, without limitation, hand or machine laundry detergentcompositions, such as laundry additive composition suitable forpre-treatment of stained fabrics and rinse added fabric softenercomposition, detergent composition for use in general household hardsurface cleaning operations, detergent compositions for hand or machinedishwashing operations. In a particular embodiment, an esterase of theinvention may be used as a detergent additive. The invention thusprovides detergent compositions comprising an esterase of the invention.Particularly, the esterase of the invention may be used as a detergentadditive in order to reduce pilling and greying effects during textilecleaning.

The present invention is also directed to methods for using an esteraseof the invention in animal feed, as well as to feed compositions andfeed additives comprising an esterase of the invention. The terms “feed”and “feed composition” refer to any compound, preparation, mixture, orcomposition suitable for, or intended for intake by an animal. Inanother particular embodiment, the esterase of the invention is used tohydrolyze proteins, and to produce hydrolysates comprising peptides.Such hydrolysates may be used as feed composition or feed additives.

It is a further object of the invention to provide a method for using anesterase of the invention in papermaking industry. More particularly,the esterase of the invention may be used to remove stickies from thepaper pulp and water pipelines of paper machines.

EXAMPLES Example 1—Construction, Expression and Purification ofEsterases

—Construction

Esterase according to the invention have been generated using theplasmidic construction. This plasmid consists in cloning a gene encodingthe esterase of SEQ ID No 1, optimized for Escherichia coli expressionbetween NdeI and XhoI restriction sites of a pET-26b(+) expressionvector (Merck Millipore, Molsheim, France). A nucleotidic sequencecoding for a PelB leader sequence has been added between SEQ ID No 1 andNdeI restriction site. Expressed fusion protein is directed to bacterialperiplasm where the PelB leader sequence is removed by a signalpeptidase giving a functional protein identical to SEQ ID No 1 but addedwith a C-terminal amino acid extension. Two site directed mutagenesiskits have been used according to the recommendations of the supplier, inorder to generate the esterase variants: QuikChange II Site-DirectedMutagenesis kit and QuikChange Lightning Multi Site-Directed fromAgilent (Santa Clara, California, USA).

—Expression and Purification of the Esterases

The strains Stellar™ (Clontech, California, USA) and E. coli BL21 (DE3)(New England Biolabs, Evry, France) have been successively employed toperform the cloning and recombinant expression in 50 mL LB-Miller mediumor ZYM auto inducible medium (Studier et al., 2005—Prot. Exp. Pur. 41,207-234). The induction in LB-Miller medium has been performed at 16°C., with 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG,Euromedex, Souffelweyersheim, France). The cultures have been stopped bycentrifugation (8000 rpm, 20 minutes at 10° C.) in an Avanti J-26 XPcentrifuge (Beckman Coulter, Brea, USA). The cells have been suspendedin 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300 mM, pH 8). Cellsuspension was then sonicated during 2 minutes with 30% of amplitude (2sec ON and 1 sec OFF cycles) by FB 705 sonicator (Fisherbrand, Illkirch,France). Then, a step of centrifugation has been realized: 30 minutes at10000 g, 10° C. in an Eppendorf centrifuge. The soluble fraction hasbeen collected and submitted to affinity chromatography. Thispurification step has been completed with Talon® Metal Affinity Resin(Clontech, CA, USA). Protein elution has been carried out with steps ofTalon buffer supplemented with imidazole. Purified protein has beendialyzed against Talon buffer then quantified using Bio-Rad proteinassay according to manufacturer instructions (Lifescience Bio-Rad,France) and stored at +4° C.

Example 2—Evaluation of the Degrading Activity of the Esterases

The degrading activity of the esterases has been determined and comparedto the activity of esterase of SEQ ID No 1.

Multiple methodologies to assess the specific activity have been used:

-   -   (1) Specific activity based upon PET hydrolysis    -   (2) Activity based upon the degradation of a polyester under        solid form    -   (3) Activity based upon PET hydrolysis in reactors above 100 mL

2.1. Specific Activity Based Upon PET Hydrolysis

100 mg of amorphous PET under powder form (prepared according to WO2017/198786 to reach a crystallinity below 20%) were weighted andintroduced in a 100 mL glass bottle. 1 mL of esterase preparationcomprising esterase of SEQ ID No 1 (as reference control) or esterase ofthe invention, prepared at 0.69 μM in Talon buffer (Tris-HCl 20 mM, NaCl0.3M, pH 8) were introduced in the glass bottle. Finally, 49 mL of 0.1 Mpotassium phosphate buffer pH 8 were added.

The depolymerization started by incubating each glass bottle at 40° C.,45° C., 50° C., 55° C., 60° C., or 70° C. and 150 rpm in a Max Q 4450incubator (Thermo Fisher Scientific, Inc. Waltham, MA, USA).

The initial rate of depolymerization reaction, in mg of equivalent TAgenerated/hour, was determined by samplings performed at different timeduring the first 24 hours and analyzed by Ultra High Performance LiquidChromatography (UHPLC). If necessary, samples were diluted in 0.1 Mpotassium phosphate buffer pH 8. Then, 150 μL of methanol and 6.5 μL ofHCl 6 N were added to 150 μL of sample or dilution. After mixing andfiltering on 0.45 μm syringe filter, samples were loaded on UHPLC tomonitor the liberation of terephthalic acid (TA), MHET and BHET.Chromatography system used was an Ultimate 3000 UHPLC system (ThermoFisher Scientific, Inc. Waltham, MA, USA) including a pump module, anautosampler, a column oven thermostated at 25° C., and an UV detector at240 nm. The column used was a Discovery® HS C18 HPLC Column (150×4.6 mm,5 μm, equipped with precolumn, Supelco, Bellefonte, USA). TA, MHET andBHET were separated using a gradient of MeOH (30% to 90%) in 1 mM ofH₂SO₄ at 1 mL/min. Injection was 20 μL of sample. TA, MHET and BHET weremeasured according to standard curves prepared from commercial TA andBHET and in house synthetized MHET in the same conditions than samples.The specific activity of PET hydrolysis (mg of equivalent TA/hour/mg ofenzyme) was determined in the linear part of the hydrolysis curve of thereaction, such curve being set up by samplings performed at differenttime during the first 72 hours. Equivalent TA corresponds to the sum ofTA measured and of TA contained in measured MHET and BHET. Saidmeasurement of equivalent TA can also be used to reflect the yield of aPET depolymerization assay at a given time.

2.2. Activity Based Upon Degradation of a Polyester Under Solid Form

20 μL of enzyme preparation was deposited in a well created in an agarplate containing PET. Preparation of agar plates was realized bysolubilizing 500 mg of PET in hexafluoro-2-propanol (HFIP) and pouringthis medium in a 250 mL aqueous solution. After HFIP evaporation at 52°C. under 140 mbar, the solution was mixed v/v with 0.2 M potassiumphosphate buffer pH 8 containing 3% agar. Around 30 mL of the mixture isused to prepare each plate and stored at 4° C.

The diameters or the surface area of the halos formed due to thepolyester degradation by wild-type esterase and variants were measuredand compared after 2 to 24 hours at 40° C., 45° C., 50° C., 60° C., 65°C. or 70° C.

2.3. Activity Based Upon PET Hydrolysis in Reactor

From 0.69 μmol to 2.07 μmol of purified esterase prepared in 80 mL of100 mM potassium phosphate buffer pH 8 were mixed with 20 g amorphousPET (prepared according to WO 2017/198786 to reach a crystallinity below20%) in a 500 mL Minibio bioreactor (Applikon Biotechnology, Delft, TheNetherlands). Temperature regulation at 40° C., 45° C., 50° C., 55° C.,65° C. or 70° C. was performed by water bath immersion and a singlemarine impeller was used to maintain constant agitation at 250 rpm. ThepH of the PET depolymerization assay was regulated at pH 8 by 6N NaOHand was assured by my-Control bio controller system (ApplikonBiotechnology, Delft, The Netherlands). Base consumption was recordedduring the assay and may be used for the characterization of the PETdepolymerization assay.

The final yield of the PET depolymerization assay was determined eitherby the determination of residual PET weight or by the determination ofequivalent TA generated, or through the base consumption. Weightdetermination of residual PET was assessed by the filtration, at the endof the reaction, of the reactional volume through a 12 to 15 μm grade 11ashless paper filter (Dutscher SAS, Brumath, France) and drying of suchretentate before weighting it. The determination of equivalent TAgenerated was realized using UHPLC methods described in 2.1, and thepercentage of hydrolysis was calculated based on the ratio of molarconcentration at a given time (TA+MHET+BHET) versus the total amount ofTA contained in the initial sample. PET depolymerization produced acidmonomers that will be neutralized with the base to be able to maintainthe pH in the reactor. The determination of equivalent TA produced wascalculating using the corresponding molar base consumption, and thepercentage of hydrolysis was calculated based on the ratio of molarconcentration at a given time of equivalent TA versus the total amountof TA contained in the initial sample.

PET depolymerization yield of the esterases (variants) of the inventionafter 24 hours are shown in Table 1 (at 50° C.) and Table 2 (at 65° C.)below. Both tables indicate the improvement of PET depolymerizationyield of the variants of the invention as compared to the PETdepolymerization yield of the esterase of SEQ ID No 1 used as reference(whose yield PET depolymerization is considered equal to 1).

The PET depolymerization yield is measured as exposed in Example 2.1.

TABLE 1 Improvement of PET depolymerization yield for an esterase of theinvention after 24 hours at 50° C. as compared to the esterase of SEQ IDNo1. Improvement of PET depolymerization yield as Variants compared toSEQ ID No1 V1: F209I + D204C + E253C + Q92G 4.4 times V2: F209I +D204C + E253C + Q92G + 9.5 times T183E V3: F209I + D204C + E253C +Q92G + 12.4 times  G135A + T168Q + T183E V4: F209I 2.4 times VariantsV1-V4 have the exact amino acid sequence as set forth in SEQ ID No1,except the combination of substitutions listed in Table 1, respectively.

Table 1 shows that the PET depolymerization yield at 50° C. of all thevariants is at least 2.4 times higher than the PET depolymerizationyield of the esterase of SEQ ID No 1.

TABLE 2 Improvement of PET depolymerization yield for an esterase of theinvention after 24 hours at 65° C. as compared to the esterase of SEQ IDNo1. Improvement of PET depolymerization yield as Variants compared toSEQ ID No1 V1: F209I + D204C + E253C + Q92G 11.0 times V2: F209I +D204C + E253C + Q92G + 25.7 times T183E V3: F209I + D204C + E253C +Q92G + 15.9 times G135A + T168Q + T183E Variants V1-V3 have the exactamino acid sequence as set forth in SEQ ID No1, except the combinationof substitutions listed in Table 2, respectively.

Table 2 shows that the PET depolymerization yield at 65° C. of all thevariants is at least 11 times higher than the PET depolymerization yieldof the esterase of SEQ ID No 1.

Specific degrading activity of esterases (variants) of the invention areshown in Table 3 below. The specific degrading activity of the esteraseof SEQ ID No 1 is used as a reference and considered as 100% specificdegrading activity. The specific degrading activity is measured asexposed in Example 2.1 at 65° C.

TABLE 3 Specific degrading activity of variants of the inventionSpecific degrading Variants activity V1: F209I + D204C + E253C + Q92G1040% V2: F209I + D204C + E253C + Q92G + T183E 2738% V3: F209I + D204C +E253C + Q92G + G135A + 1958% T168Q + T183E Variants V1-V3 have the exactamino acid sequence as set forth in SEQ ID No1, except the combinationof substitutions listed in Table 3, respectively.

Example 3—Evaluation of the Thermostability of Esterases of theInvention

The thermostability of esterases of the invention has been determinedand compared to the thermostability of the esterase of SEQ ID No 1.

Different methodologies have been used to estimate thermostability:

-   -   (1) Circular dichroism of proteins in solution;    -   (2) Residual esterase activity after protein incubation in given        conditions of temperatures, times and buffers;    -   (3) Residual polyester's depolymerization activity after protein        incubation in given conditions of temperatures, times and        buffers;    -   (4) Ability to degrade a solid polyester compound (such as PET        or PBAT or analogues) dispersed in an agar plate, after protein        incubation in given conditions of temperatures, times and        buffers;    -   (5) Ability to perform multiple rounds of polyester's        depolymerization assays in given conditions of temperatures,        buffers, protein concentrations and polyester concentrations;    -   (6) Differential Scanning Fluorimetry (DSF);

Details on the protocol of such methods are given below.

3.1 Circular Dichroism

Circular dichroism (CD) has been performed with a Jasco 815 device(Easton, USA) to compare the melting temperature (Tm) of the esterase ofSEQ ID No 1 with the Tm of the esterases of the invention. Technically4004, protein sample was prepared at 0.5 mg/mL in Talon buffer and usedfor CD. A first scan from 280 to 190 nm was realized to determine twomaxima intensities of CD corresponding to the correct folding of theprotein. A second scan was then performed from 25° C. to 110° C., atlength waves corresponding to such maximal intensities and providingspecific curves (sigmoid 3 parameters y=a/(1−e{circumflex over( )}((x−x0)/b))) that were analyzed by Sigmaplot version 11.0 software,the Tm is determined when x=x0. The T_(m) obtained reflects thethermostability of the given protein. The higher the T_(m) is, the morestable the variant is at high temperature.

3.2 Residual Esterase Activity

1 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQID No 1 or of an esterase of the invention was incubated at differenttemperatures (40, 50, 60, 65, 70, 75, 80 and 90° C.) up to 10 days.Regularly, a sample, was taken, diluted 1 to 500 times in a 0.1Mpotassium phosphate buffer pH 8.0 and para nitro phenol-butyrate (pNP-B)assay was realized. 20 μL of sample are mixed with 175 μL of 0.1Mpotassium phosphate buffer pH 8.0 and 5 μL of pNP-B solution in2-methyl-2 butanol (40 mM). Enzymatic reaction was performed at 30° C.under agitation, during 15 minutes and absorbance at 405 nm was acquiredby microplate spectrophotometer (Versamax, Molecular Devices, Sunnyvale,CA, USA). Activity of pNP-B hydrolysis (initial velocity expressed inμmol of pNPB/min) was determined using a standard curve for theliberated para nitro phenol in the linear part of the hydrolysis curve.

3.3 Residual Polyester Depolymerizing Activity

10 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQID No 1 and of an esterase of the invention respectively were incubatedat different temperatures (40° C., 50° C., 60° C., 65° C., 70° C., 75°C., 80° C. and 90° C.) up to 30 days. Regularly, a 1 mL sample wastaken, and transferred into a bottle containing 100 mg of amorphous PET(prepared according to WO 2017/198786 to reach a crystallinity below20%) micronized at 250-500 μm and 49 mL of 0.1M potassium phosphatebuffer pH 8.0 and incubated at 50° C., 55° C., 60° C., 65° C. or 70° C.150 μL of buffer were sampled regularly. When required, samples werediluted in 0.1 M potassium phosphate buffer pH 8. Then, 150 μL ofmethanol and 6.5 μL of HCl 6 N were added to 150 μL of sample ordilution. After mixing and filtering on 0.45 μm syringe filter, sampleswere loaded on UHPLC to monitor the liberation of terephthalic acid(TA), MHET and BHET. Chromatography system used was an Ultimate 3000UHPLC system (Thermo Fisher Scientific, Inc. Waltham, MA, USA) includinga pump module, an autosampler, a column oven thermostated at 25° C., andan UV detector at 240 nm. The column used was a Discovery® HS C18 HPLCColumn (150×4.6 mm, 5 μm, equipped with precolumn, Supelco, Bellefonte,USA). TA, MHET and BHET were separated using a gradient of MeOH (30% to90%) in 1 mM of H₂SO₄ at 1 mL/min. Injection was 20 μL of sample. TA,MHET and BHET were measured according to standard curves prepared fromcommercial TA and BHET and in house synthetized MHET in the sameconditions than samples. Activity of PET hydrolysis (μmol of PEThydrolysed/min or mg of equivalent TA produced/hour) was determined inthe linear part of the hydrolysis curve, such curve being set up bysamplings performed at different time during the first 24 hours.Equivalent TA corresponds to the sum of TA measured and of TA containedin measured MHET and BHET.

3.4 Degradation of a Polyester Under Solid Form

1 mL of a solution of 40 mg/L (in Talon buffer) of the esterase of SEQID No 1 and of an esterase of the invention respectively were incubatedat different temperatures (40° C., 50° C., 60° C., 65° C., 70° C., 75°C., 80° C. and 90° C.) up to 30 days. Regularly, 20 μL of enzymepreparation was 30 deposited in a well created in an agar platecontaining PET. Preparation of agar plates containing PET was realizedby solubilizing 500 mg of PET in hexafluoro-2-propanol (HFIP), andpouring this medium in a 250 mL aqueous solution. After HFIP evaporationat 52° C. under 140 mbar, the solution was mixed v/v with 0.2 Mpotassium phosphate buffer pH 8 containing 3% agar. Around 30 mL of themixture is used to prepare each omnitray and stored at 4° C.

The diameter or the surface area of the halos formed due to thepolyester degradation by wild-type esterase and variants of theinvention were measured and compared after 2 to 24 hours at 50° C., 55°C., 60° C., 65° C. or 70° C. The half-life of the enzyme at a giventemperature corresponds to the time required to decrease by a 2-foldfactor the diameter of the halo.

3.5 Multiple Rounds of Polyester's Depolymerization

The ability of the esterase to perform successive rounds of polyester'sdepolymerization assays was evaluated in an enzymatic reactor. A Minibio500 bioreactor (Applikon Biotechnology B.V., Delft, The Netherlands) wasstarted with 3 g of amorphous PET (prepared according to WO 2017/198786to reach a crystallinity below 20%) and 100 mL of 10 mM potassiumphosphate buffer pH 8 containing 3 mg of esterase. Agitation was set at250 rpm using a marine impeller. Bioreactor was thermostated at 50° C.,55° C., 60° C., 65° C. or 70° C. by immersion in an external water bath.pH was regulated at 8 by addition of KOH at 3 M. The differentparameters (pH, temperature, agitation, addition of base) were monitoredthanks to BioXpert software V2.95. 1.8 g of amorphous PET (preparedaccording to WO 2017/198786 to reach a crystallinity below 20%) wereadded every 20 h. 500 μL of reaction medium was sampled regularly.

Amount of TA, MHET and BHET was determined by HPLC, as described inexample 2.3. Amount of EG was determined using an Aminex HPX-87K column(Bio-Rad Laboratories, Inc, Hercules, California, United States)thermostated at 65° C. Eluent was K₂HPO₄ 5 mM at 0.6 mL·min⁻¹. Injectionwas 20 μL. Ethylene glycol was monitored using refractometer.

The percentages of hydrolysis were calculated based on the ratio ofmolar concentration at a given time (TA+MHET+BHET) versus the totalamount of TA contained in the initial sample, or based on the ratio ofmolar concentration at a given time (EG+MHET+2×BHET) versus the totalamount of EG contained in the initial sample. Rate of degradation iscalculated in mg of total liberated TA per hour or in mg of total EG perhour.

Half-life of enzyme was evaluated as the incubation time required toobtain a loss of 50% of the degradation rate.

3.6 Differential Scanning Fluorimetry (DSF)

DSF was used to evaluate the thermostability of the wild-type protein(SEQ ID No 1) and variants thereof by determining their meltingtemperature (Tm), temperature at which half of the protein population isunfolded. Protein samples were prepared at a concentration of 14 andstored in buffer A consisting of 20 mM Tris HCl pH 8.0, 300 mM NaCl. TheSYPRO orange dye 5000× stock solution in DMSO was first diluted to 250×in water. Protein samples were loaded onto a white clear 96-well PCRplate (Bio-Rad cat #HSP9601) with each well containing a final volume of25 μl. The final concentration of protein and SYPRO Orange dye in eachwell were 5 μM (0.14 mg/ml) and 10× respectively. Loaded volumes perwell were as follow: 15 μL of buffer A, 9 μL of the 14 μM proteinsolution and 1 μL of the 250× Sypro Orange diluted solution. The PCRplates were then sealed with optical quality sealing tape and spun at2000 rpm for 1 min at room temperature. DSF experiments were thencarried out using a CFX96 real-time PCR system set to use the 450/490excitation and 560/580 emission filters. The samples were heated from 25to 100° C. at the rate of 0.3° C./second. A single fluorescencemeasurement was taken every 0.03 second. Melting temperatures weredetermined from the peak(s) of the first derivatives of the meltingcurve using the Bio-Rad CFX Manager software.

Esterase of SEQ ID No 1 and esterases of the invention were thencompared based on their Tm values. Due to high reproducibility betweenexperiments on the same protein from different productions, a ΔTm of0.8° C. was considered as significant to compare variants. Tm valuescorrespond to the average of at least 3 measurements.

Tm of the esterase of SEQ ID No 1 is evaluated equal to 68.0° C.+/−0.2°C. as exposed in Example 3.6.

Thermostabilities of esterase variants of the invention are shown inTable 4 below, expressed in Tm values and evaluated according to Example3.6. The gain of Tm as compared to the esterase of SEQ ID No 1 isindicated in brackets.

TABLE 4 Tm of the esterase of the invention Variants Tm V1: F209I +D204C + E253C + Q92G 87.6° C. (+19.6° C.) V2: F209I + D204C + E253C +Q92G + T183E 90.0° C. (+22.0° C.) V3: F209I + D204C + E253C + Q92G +82.6° C. (+14.6° C.) G135A + T168Q + T183E Variants V1-V3 have the exactamino acid sequence as set forth in SEQ ID No1, except the combinationof substitutions listed in Table 4, respectively.

1-36. (canceled)
 37. An esterase which (i) has at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identity to the full length amino acidsequence set forth in SEQ ID NO: 1, and (ii) has at least one amino acidsubstitution selected from F209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H,W69R, T89R/F, D94S, S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y,S197P, E202M, G205C/K, T207L, P211A, N212D/Q, K216P, K220E, Q238T,L240A, P242K, G243Y, P244N, G247A/D/E/H/S, G250C/Y and P260S wherein thepositions are numbered by reference to the amino acid sequence set forthin SEQ ID NO: 1, (iii) has a polyester degrading activity, and (iv) hasan increased thermostability and/or an increased degrading activitycompared to the esterase of SEQ ID NO:
 1. 38. The esterase according toclaim 37, wherein said esterase comprises at least one substitutionselected from the group consisting of T168Q, F209I/G/H/L/R/T andN212D/Q.
 39. The esterase according to claim 37, wherein said esterasecomprises at least one substitution selected from F209I/G/H/L/R/T andT168Q.
 40. The esterase according to claim 37, wherein said esterasecomprises at least one substitution selected from F209I/G/H/L/R/T. 41.The esterase according to claim 37, wherein said esterase furthercomprises at least one substitution selected from T11N, D12H, S23P,T50E, A53L, Y60F/M, T61M/V, T63N, A65T, T88S, T89Q, L90W/F, Q92G/P,M107L, S121R, A125G, L152Q, M127V, G135A, S136T, P151A, L157E/G/N/Q/W/T,K159T, T177N/H/Q, T183E, D204C/K/R, T207D, F209W/S/A, A210T, N212M,I213F, K216N, Q238D, D246Y/C/E/P, E253C and D174R.
 42. The esteraseaccording to claim 41, wherein said esterase further comprises at leastone substitution selected from Q92G/P, G135A, T183E, D204C/K/R andE253C.
 43. The esterase according to claim 37, wherein said esterasefurther comprises at least one substitution selected from Q92G/P, G135A,T183E, D204C/K/R, F209W/S/A and E253C.
 44. The esterase according toclaim 37, wherein said esterase further comprises at least thecombination of substitutions D204C+E253C.
 45. The esterase according toclaim 37, wherein said esterase comprises a substitution selected fromF209I/A/G/H/L/R/S/T and at least one, two or three substitution(s)selected from T61M/V, A65T, Q92G/P, G135A, T168Q, T177N/H/Q, T183E,D204C/K/R, N212D/Q/M and E253C.
 46. The esterase according to claim 37,wherein said esterase comprises at least four amino acid substitutionsat positions selected from F209, T61, A65, Q92, G135, T168, T177, T183,D204, N212 and E253.
 47. The esterase according to claim 37, whereinsaid esterase comprises at least a combination of substitutions atpositions selected from the group consisting of F209+D204+E253,F209+D204+E253+Q92, F209+D204+E253+N212, F209+D204+E253+Q92+G135+T168,F209+D204+E253+Q92+G135+T168+T183, F209+D204+E253+Q92+T183, orF209+D204+E253+Q92+T168+T183.
 48. The esterase according to claim 37,wherein said esterase comprises at least a combination of substitutionat positions F209+D204+E253+Q92.
 49. The esterase according to claim 37,wherein said esterase comprises at least a combination of substitutionsselected from F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+N212D/M/Q,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+G135A+T168Q,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+G135A+T168Q+T183E,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+T183E,F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C+Q92G/P+T168Q+T183E.
 50. Theesterase according to claim 49, wherein the amino acid sequence of saidesterase consists of the amino acid sequence as set forth in SEQ ID NO:1 with a combination of substitutions, as compared to SEQ ID NO: 1,selected from F209I/A/G/H/L/R/S/T/W+D204C+E253C,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P,F209I/A/G/H/L/R/S/T/W+D204C+E253C+N212D/M/Q,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+G135A+T168Q,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+G135A+T168Q+T183E,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+T183E,F209I/A/G/H/L/R/S/T/W+D204C+E253C+Q92G/P+T168Q+T183E.
 51. The esteraseaccording to claim 37, wherein said esterase has the amino acid sequenceset forth in SEQ ID NO: 1 with one to thirty-one amino acidsubstitutions, as compared to SEQ ID NO: 1, selected from the groupconsisting of F209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F,D94S, S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M,G205C/K, T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A, P242K,G243Y, P244N, G247A/D/E/H/S, G250C/Y and P260S.
 52. The esteraseaccording to claim 37, wherein the amino acid sequence of said esteraseconsists of the amino acid sequence as set forth in SEQ ID NO: 1 with asingle amino acid substitution, as compared to SEQ ID NO: 1, selectedfrom F209I/G/H/L/R/T, D12F/Y/R, T50P, T63Q, S66H, W69R, T89R/F, D94S,S121W, T153A, N158Q, T168Q, P180E, A182R, F188I/Y, S197P, E202M,G205C/K, T207L, P211A, N212D/Q, K216P, K220E, Q238T, L240A, P242K,G243Y, P244N, G247A/D/E/H/S, G250C/Y and P260S.
 53. The esteraseaccording to claim 52, wherein the single substitution is selected fromF209I/G/H/L/R/T.
 54. The esterase according to claim 37, wherein saidesterase comprises at least one amino acid residue selected from D176,H208, S130, M131, C241, C259, G59, H129, G132, W155, I171, I178, P214,D174, as set forth in SEQ ID NO:
 1. 55. The esterase according to claim37, wherein said esterase comprises at least one amino acid selectedfrom G59, H129, G132, W155, I171, I178 and P214 as set forth in SEQ IDNO:
 1. 56. The esterase according to claim 37, wherein said esteraseexhibits both an increased thermostability and an increased degradingactivity compared to the esterase of SEQ ID NO:
 1. 57. An esterase which(i) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity tothe full length amino acid sequence set forth in SEQ ID NO: 1, and (ii)has at least three amino acid substitutions at positions selected fromF209, T61, A65, Q92, G135, T168, T177, T183, D204, N212 and E253,wherein the positions are numbered by reference to the amino acidsequence set forth in SEQ ID NO: 1, (iii) has a polyester degradingactivity, and (iv) has an increased thermostability and/or an increaseddegrading activity compared to the esterase of SEQ ID NO:
 1. 58. Theesterase according to claim 57, wherein said esterase comprises at leastthree amino acid substitutions selected from F209I/A/G/H/L/R/S/T/W,Q92G/P, G135A, T168Q, T183E, D204C/K/R, and E253C.
 59. The esteraseaccording to claim 57, wherein said esterase comprises at least acombination of substitutions at positions F209+D204+E253.
 60. Theesterase according to claim 59, wherein said esterase further compriseat least one substitution at a position selected from T61, A65, Q92,G135, T168, T177, T183 and N212.
 61. The esterase according to claim 59,wherein the esterase comprises at least a combination of substitutionselected from F209I/A/G/H/L/R/S/T/W+D204C/K/R+E253C and at least onesubstitution selected from T61M/V, A65T, Q92G/P, G135A, T168Q,T177N/H/Q, T183E and N212D/Q/M.
 62. A nucleic acid encoding an esteraseaccording to claim 37, a nucleic acid encoding said esterase, a vectoror expression cassette comprising said nucleic acid or a host cellcomprising said nucleic acid.
 63. A composition comprising an esteraseaccording to claim 37 or a host cell comprising a nucleic acid encodingsaid esterase.
 64. A method of degrading a polyester comprising (a)contacting the polyester with an esterase according to claim 37 or ahost cell comprising a nucleic acid encoding said esterase; and,optionally (b) recovering monomers and/or oligomers.
 65. A polyestercontaining material containing an esterase according to claim 37 or ahost cell comprising a nucleic acid encoding said esterase.
 66. Adetergent composition comprising the esterase according to claim 37.